Clathrin-coated vesicles (CCVs) are involved in receptor-mediated endocytosis and in the transport of lysosomal and vacuolar proteins from the trans-Golgi network (TGN). Clathrin and the clathrin-associated adaptor (or assembly) protein complexes (APs) are the main protein components of the coat surrounding the cytoplasmic face of coated vesicles (Kirchhausen, T. et al. (1991) J. Biol. Chem. 266:11153-11157). AP-1 and AP-2 are found in clathrin-coated structures which bud from Golgi and plasma membranes, respectively.
APs are believed to interact with the cytoplasmic tails of membrane proteins, and to assist in the selection and concentration of the membrane proteins into CCVs (Kirchhausen, et al., supra). APs are heterotetrameric structures which contain two large chains of approximately 100 kdal designated .beta.'-adaptin and .gamma.-adaptin in AP-1 complexes, and .alpha.-adaptin and .beta.-adaptin in AP-2 complexes; a medium chain of approximately 50 kdal, designated AP47 in AP-1 and AP-50 in AP-2; and a small chain of approximately 18 kdal, designated AP19 in AP-1 and AP17 in AP-2. AP proteins have been cloned from a variety of sources including mouse and yeast (Kirchhausen, et al., supra; Nakai, M. et al. (1993) Biochim. Biophys. Acta 1174:282-284).
In receptor-mediated endocytosis, CCVs capture plasma membrane receptor proteins through the interaction of the receptor proteins with AP-2 complex. This interaction enriches for receptors which are destined for internalization and excludes proteins which remain at the cell surface. Some receptors, such as the low-density lipoprotein (LDL) receptor, are constitutively included within the vesicle, while other receptors, such as insulin and epidermal growth factor receptors, are mobilized only in response to a ligand (Scheckman, R. et al. (1996) Science 271:1526-1533).
In the TGN, CCVs capture receptors involved in traffic of lysosomal and vacuolar proteins. Recruitment in this location is mediated by the AP-1 adaptor complex (.beta.'- and .gamma.-adaptins, AP47, and AP19). CCVs which bud from the TGN incorporate mannose-6-phosphate receptors, which bind newly synthesized lysosomal enzymes on the lumenal side of the membrane (Seaman, M. N. J. et al. (1996) J. Biol. Chem. 271:25446-25451). Enzymes are thus recruited into CCVs and are delivered to prelysosomal compartments.
Lysosomes contain numerous enzymes, including proteases, lipases, and glucosidases, which digest cellular constituents down to their basic building blocks, amino acids, fatty acids, and sugars. Lysosomes thus function as an intracellular digestive system. They are necessary for the normal digestion of cellular nutrients, intracellular debris, and potentially harmful extracellular substances which must be removed from the body. Disruption of the lysosomal membrane results in a release of lysosomal enzymes and leads to tissue damage. Tissue destruction associated with inflammation is due in part to the release of lysosomal enzymes from phagocytes. Other aspects of the inflammatory response attributed to released lysosomal components include increased vascular permeability, breakdown of connective tissue, and activation of the complement and kinin systems.
Lysosomal storage diseases are attributed to defects in or lack of one or more lysosomal enzymes. For instance, a deficiency in glucocerebrosidease results in Gaucher's disease, an accumulation of glucocerebroside in macrophage lysosomes. The lack of lysosomal alpha-1,4,-glucosidase leads to Pome's disease, an accumulation of glycogen in the lysosomes. A deficiency or absence of lysosomal hexosaminidase results in Tay-Sachs disease, a neurodegenerative disorder arising from the accumulation of the lipid GM2 ganglioside in lysosomes of neuronal cells. The accumulation of spingomyelin in neuronal cells leads to neurological disorders; for instance, a deficiency in sphingomyelinase leads to Niemann-Pick disease. Furthermore, excessive accumulation of undigested molecules in the lysosome may generate abnormal immune responses which lead to autoimmune disorders.
Polynucleotides encoding a clathrin-associated protein and the molecules themselves provide a means to investigate CCV trafficking under normal and disease conditions. Discovery of a clathrin-associated protein satisfies a need in the art by providing new compositions useful in diagnosing and treating disorders associated with abnormal vesicle trafficking.